P152: Redirecting metabolic flux from lactate to ethanol in Lactobacillus casei 12A

Lactobacillus casei 12A has been selected as the biofuels parental strain based upon its alcohol tolerance (>10% ethanol), carbohydrate utilization, and relatively high transformation efficiency. This organism metabolizes hexoses through the Embden-Meyerhof-Parnas pathway and converts pyruvate to lactate via a variety of different enzymes; including four L- lactate dehydrogenases (L-Ldh), one D-Ldh, and one D-hydroxyisocaproate dehydrogenase (D-Hic). We are pursuing two strategies concurrently to redirect L. casei 12A fermentation to ethanol. The first strategy involves inactivation of Ldhs and D-Hic that consume pyruvate under anaerobic conditions without producing ethanol. To date, this approach has been used to inactivate L-ldh1, L-ldh2, and D-hic as well as construct the L-ldh1/L-ldh2, double mutant. The highest level of ethanol formation was observed with the ΔL-ldh1/ΔL-ldh2 double mutant, which produces ethanol as 14% of its metabolic end products. Our second strategy for increasing flux to ethanol involves expression of ethanol producing enzymes. A codon optimized “PET” cassette comprised of the Zymomonas mobilis genes encoding pyruvate decarboxylase (Pdc) and alcohol dehydrogenase (Adh2) was constructed, and placed under the control of the L. casei 12A pgm promoter and kdgR transcriptional terminator. When this construct was introduced into L. casei 12A ethanol made up 61% of metabolic end products formed. When introduced into L. casei 12A (ΔL-ldh1) ethanol was the dominant product observed (91% of metabolic end productions). Results from this analysis indicate that the two approaches are complementary and demonstrate that redirecting metabolic flux in L. casei from lactate to an alcohol can be readily achieved.